Previous Article | Next Article 
Journal of Virology, February 1999, p. 1165-1174, Vol. 73, No. 2
First Department of Internal Medicine, Kanazawa
University, Kanazawa, Japan1;
Department
of Microbiology and Immunology, The University of Texas Medical Branch
at Galveston, Galveston, Texas 77555-10192; and
Department of Medicine, The University of North Carolina at
Chapel Hill, Chapel Hill, North Carolina 27599-70303
Received 9 July 1998/Accepted 1 November 1998
Hepatitis C virus (HCV) initiates translation of its
polyprotein under the control of an internal ribosome entry site (IRES) that comprises most of the 341-nucleotide (nt) 5' nontranslated RNA
(5'NTR). A comparative analysis of related flaviviral sequences suggested that an RNA segment for which secondary structure was previously ill defined (domain II, nt 44 to 118) forms a conserved stem-loop that is located at the 5' border of the HCV IRES and thus may
function in viral translation. This prediction was tested by a
mutational analysis of putative helical structures that examined the
impact of both covariant and noncovariant nucleotide substitutions on
IRES activity in vivo and in vitro. Results of these experiments provide support for predicted base pair interactions between nt 44 to
52 and 111 to 118 and between nt 65 to 70 and 97 to 102 of the HCV
5'NTR. Substitutions at either nt 45 and 46 or nt 116 and 117 resulted
in reciprocal changes in V1 nuclease cleavage patterns within the
opposing strand of the putative helix, consistent with the predicted
base pair interactions. IRES activity was highly dependent on
maintenance of the stem-loop II structure but relatively tolerant of
covariant nucleotide substitutions within predicted helical segments.
Sequence alignments suggested that the deduced domain II structure is
conserved within the IRESs of pestiviruses as well as the novel
flavivirus GB virus B. Despite marked differences in primary nucleotide
sequence within conserved helical segments, the sequences of the
intervening single-stranded loop segments are highly conserved in these
different viruses. This suggests that these segments of the viral RNA
may interact with elements of the host translational machinery that are
broadly conserved among different mammalian species.
Hepatitis C virus (HCV) is a
positive-strand, enveloped RNA virus that is classified within
the genus Hepacivirus of the family Flaviviridae
(3). This virus establishes a persistent infection in most
infected individuals, potentially leading to the development of
chronic hepatitis, cirrhosis, or hepatocellular carcinoma (3, 12). It is thus a major cause of liver-specific morbidity and mortality in human populations. HCV isolates recovered from
different patients demonstrate considerable genetic diversity (4,
21), and there is extensive quasispecies variation among HCV
sequences recovered from individual infected patients
(10, 31). However, the nucleotide sequence of the
5' nontranslated RNA (5'NTR) is relatively well
conserved among different genotypes of HCV. This conservation of
primary structure likely reflects requirements for
higher-ordered RNA structures that control translation and/or replication of the viral genome.
A number of previous studies have demonstrated the presence of an
internal ribosome entry site (IRES) within the 5'NTR of HCV that
directs the cap-independent initiation of virus translation (6,
11, 16, 17, 27, 30). Thus, the initiation of translation on HCV
RNA occurs by a mechanism that is different from the cap-dependent
translation initiation of yellow fever virus and other members of the
genus Flavivirus (25). As an entity involved in
highly specific macromolecular interactions (14), the IRES
is a reasonable target for antiviral drug development. A detailed
understanding of its structure is likely to contribute to such efforts.
Functional and structural studies of the HCV IRES have been carried out
in a number of laboratories (1, 6, 9, 11, 14-19, 27-30).
Most of these studies have drawn on a model of the secondary structure
of the 5'NTR of HCV that was proposed by Brown et al. in 1992 (2). This model was modified by Wang et al. in 1995 (28) following the demonstration of a pseudoknot within the
5'NTR that is required for translation, and it was further refined by
Smith et al. (24) in 1995 and Honda et al. (9) in
1996. To a considerable extent, the model is based on a comparative analysis of the sequences of multiple strains of HCV and members of the
genus Pestivirus (bovine viral diarrhea virus [BVDV] and hog cholera virus [HoCV]) (2). Although the model has been validated by both physical probing of RNA structure and mutational analysis of IRES function, the assignment of structure has been problematic within the 5' half of the 5'NTR (domain II). This is due
the fact that there is considerable divergence of the nucleotide sequences of different genera of the family Flaviviridae in
this region, despite strong conservation of this sequence among
different HCV strains. This has made covariant sequence analysis
difficult. Furthermore, there have been few attempts at mutational
analysis of this part of the IRES structure. Thus, it is not surprising that quite different structures have been proposed in the past for
these regions of the HCV and pestiviral 5'NTRs (2).
We recently presented a significantly different prediction of the
secondary structure of this segment of the HCV 5'NTR based on a
comparison of the HCV sequence with that of a newly discovered, hepatotropic mammalian virus, GB virus B (GBV-B) (9, 23). Unlike the more distantly related GB viruses A and C (22),
GBV-B has a number of features in common with HCV, including many
aspects of the secondary structure of its 5'NTR (9, 23). In
the revised structural model, domain II was suggested to form a complex
structure with two hairpins, extending from nucleotides (nt) 44 to 117 and containing a large, internal single-stranded bulge loop (nt 103 to
114) (Fig. 1A) (9). These
latter structures were supported by previous nuclease mapping studies
(2). However, computer-based, thermodynamic predictions of
this domain, constrained with respect to predicted base pair
interactions at its base, suggest that a more stable structure may be
formed by alternative interactions between bases predicted previously
to form hairpin IIa and those in the opposing bulge loop (Fig. 1B).
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A Phylogenetically Conserved Stem-Loop Structure at the
5' Border of the Internal Ribosome Entry Site of Hepatitis C Virus
Is Required for Cap-Independent Viral Translation
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
View larger version (18K):
[in a new window]
FIG. 1.
Alternative predictions of secondary RNA structure
within domain II of the 5'NTR of HCV (N strain, genotype Ib). (A)
Structure suggested by a recent comparison of the HCV and GBV-B
sequences, comprising a complex stem-loop extending from nt 44 to 118, with two internal hairpin loops, IIa (nt 50 to 64) and IIb (nt 65 to
102), and a large, internal single-stranded bulge loop (nt 103 to 114)
(9). Predicted 
G = 
8.6 kcal/mol. (B)
Structure predicted by MFOLD when folding is constrained to include
base pairing between nt 44 to 47 and nt 115 to 118, as suggested by the
GBV-B sequence comparison. G = 18.2 kcal/mol.
Here, we describe a mutational analysis of this segment of the 5'NTR of HCV. We present data supporting the existence of the structure shown in Fig. 1B and show that its integrity is essential to IRES-directed translation of the HCV open reading frame. An analysis of the nucleotide sequences of the pestiviruses and GBV-B indicates that this predicted structure is broadly conserved among those members of Flaviviridae that initiate translation of the polyprotein by internal ribosome entry.
| |
MATERIALS AND METHODS |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
Plasmids.
Plasmid pCAT-N-CLuc contains a bicistronic, T7
transcriptional unit consisting of the chloramphenicol
acetyltransferase (CAT) sequence fused at its 3' end to the 5'NTR of
the N strain of HCV (7), followed by 66 nt of the HCV open
reading frame fused in frame with the luciferase sequence
(8). pCAT-N-CE1 is a similar bicistronic plasmid in which
the complete HCV capsid and 5' E1 coding sequences represent the entire
second cistron (8).
E1-related
mutants (see Results) that contain nucleotide substitutions within
segments of the domain II sequence that are predicted to participate in
helix formation. To construct these plasmids, a shuttle vector,
pSP73(1-279), was made by subcloning the
SalI/StuI fragment (HCV nt 1 to 279) of
pCAT-N-CLuc into the cloning vector pSP73 (Promega). Site-directed
mutagenesis was carried out by a standard PCR-based strategy using
selected oligonucleotide primers and Pfu (Pyrococcus
furiosus) DNA polymerase. The mutated sequences were subsequently
inserted into the pCAT-N-CLuc plasmid or used to replace homologous
sequence in plasmid pCAT-N-CE1. Alternatively, some mutations were
constructed directly in pCAT-N-CLuc by using a QuickChange
site-directed mutagenesis kit (Stratagene). Plasmid DNAs were purified
on Qiagen-tip 500 columns (Qiagen, Chatsworth, Calif.). The sequences
of all PCR-manipulated regions and the presence of expected mutations
were confirmed by DNA sequencing.
Cells. Huh-T7 cells (20), derived from human hepatocellular carcinoma (Huh-7) cells, are stably transformed and constitutively express bacteriophage T7 RNA polymerase. BT7-H cells (32), derived from African green monkey kidney (BS-C-1) cells, similarly produce T7 RNA polymerase constitutively. Cell cultures were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin, streptomycin, and 400 µg (active compound) of geneticin (Gibco-BRL) per ml.
In vitro transcription and translation. In vitro transcription-translation reactions were carried out with the Promega TNT coupled reticulocyte lysate system as specified by the manufacturer. Reaction mixtures (25 µl) contained 20 U of RNasin (Promega), 20 µCi of [35S]methionine (DuPont NEN), and 0.5 µg of plasmid template. After incubation at 30°C for 2 h, 3-µl aliquots of the translation products were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% polyacrylamide-6 M urea gels, which were then subjected to autoradiography. The quantities of reporter proteins produced in these reactions were determined by enzymatic assay (see below) or by PhosphorImager analysis (Molecular Dynamics).
In vivo analysis of HCV translation in Huh-T7 cells. Huh-T7 cells were seeded into six-well tissue culture plates 24 h before transfection. Since these cells have a relatively low level of endogenous T7 polymerase expression, they were infected when 90% confluent with a recombinant vaccinia virus, vTF7-3, that expresses the polymerase (5). The inoculum was in 500 µl of Opti-MEM (Gibco-BRL) at a multiplicity of infection of 5. After a 1-h incubation at 37°C, the inoculum was removed and replaced with a mixture containing 2 µg of plasmid DNA and 15 µl of Lipofectin (Gibco-BRL) in 200 µl of Opti-MEM at 37°C, followed 15 min later by an additional 800 µl of Opti-MEM (11). Cells were subsequently incubated at 37°C in a 5% CO2 atmosphere for 18 to 24 h. At harvest, 1 ml of 10 mM EDTA-phosphate-buffered saline was added to the cells, which were then incubated at 37°C for 10 min, collected, and divided into two equal aliquots. Following low-speed centrifugation, cells were resuspended in 500 µl of 1× lysis buffer (Promega) for luciferase assay or 500 µl of 0.25 M Tris-HCl for CAT assay. For the luciferase assay (Promega), 20-µl aliquots were assayed for light production. For the CAT assay, cells were lysed by freeze-thawing and lysates were further diluted to 1:500. CAT activity was determined by a phase extraction assay, which measures butyrylated [14C]chloramphenicol products by liquid scintillation counting, following xylene extraction (pCAT reporter gene system; Promega).
In vivo analysis of HCV translation in BT7-H cells. BT7-H cells that constitutively express the T7 polymerase were seeded into six-well tissue culture plates and allowed to reach 90 to 95% confluency. Transfection was performed by the addition of a mixture containing 2 µg of plasmid DNA, 6 µl of FuGENE 6 transfection reagent (Boehringer Mannheim), and 92 µl of Opti-MEM (Gibco-BRL) and then added directly to cells in regular maintenance medium containing 10% fetal calf serum. Cells were incubated at 37°C in a 5% CO2 atmosphere for 24 h, after which cells were harvested and lysates were assayed for luciferase and CAT activity as described above.
Computer-based prediction of domain II RNA folding. Thermodynamic predictions of RNA secondary structure at 37°C were developed by using the MFOLD 3.0 program with Turner energies and selected constraints on base pairing as indicated. The program was run on the server supporting the Zuker web site at the University of Washington (http://www.ibc.wustl.edu/~zuker/mfold).
Nuclease mapping of RNA secondary structure. Synthetic HCV RNA was generated by in vitro transcription of AatII-linearized plasmid pCAT-N-CLuc with T7 RNA polymerase. Transcripts were heated to 65°C for 3 min and allowed to cool slowly to 4°C in the presence of 10 mM MgCl2-10 mM Tris (pH 7.6). Enzymatic modification of 1 µg of RNA was carried out with RNase V1 (Pharmacia) or RNase S1 (Pharmacia) at room temperature in the presence of 20 µg of carrier tRNA and 10 mM MgCl2-10 mM Tris (and 1 mM ZnSO4 for S1 nuclease) at pH 7.6 in a total volume of 40 µl for 10 min. Reactions were stopped by the addition of excess tRNA. The modified RNA was ethanol precipitated and analyzed by primer extension with a 32P-labeled, negative-strand HCV primer and 3 U of avian myeloblastosis virus reverse transcriptase (Life Sciences) at 42°C for 30 min. The primers used in these reactions were complementary to HCV sequences commencing at nt 110 and 190 of the viral genome. Reaction products were separated on a 6% polyacrylamide gel in parallel with dideoxynucleotide sequencing reactions of unmodified RNA.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
Alternative predictions of the secondary structure of
domain II of the HCV IRES.
The recent determination of the
nucleotide sequence of GBV-B, a novel hepatotropic flavivirus
(23), provided an additional HCV-related 5'NTR sequence
which could be used in a search for covariant nucleotide changes
indicative of conserved secondary RNA structure. This analysis led us
to propose a significant revision of the structure of domain II of the
5'NTR of HCV, a segment of the 5'NTR which is known to contribute to
IRES function (Fig. 1A) (2, 9). The most significant
difference between this predicted structure (9) and the
previous prediction (2) was the base pairing of nt 115 to
118 with nt 44 to 47, to form a putative helix located at the base of
the structure. This potential base pairing is preserved in the sequence
of GBV-B (with conservation of the primary sequence within the 3'
segment of the helix), unlike the previously predicted base pair
arrangement in this region of the 5'NTR (9). We subsequently
used the MFOLD program to predict an optimal structure for the
intervening sequence, constraining the fold to include the nt
44-47/115-118 interaction. The result (Fig. 1B) was a structure in
which the previously predicted hairpin IIa was no longer present and in
which an extension of the basal helix included pairing between nt 49 to
52 and nt 111 to 114. This second structure is significantly more
stable than that shown in Fig. 1A (G = 16.8 versus
8.6 kcal/mol). However, since the involved sequences are highly
conserved among HCV strains (24), comparative sequence
analysis provides no clues as to which structure is correct.
Furthermore, with few exceptions, both structures conform to previously
identified sites of cleavage by single- and double-strand-specific
nucleases (2).
Mutations which destabilize base pairing between nt 44 to 47 and nt 115 to 118 decrease IRES activity. We carried out a mutational analysis to verify predicted helical segments that are common to both of the structures shown in Fig. 1, as well as to distinguish between the two predictions. We created mutations within the background of a dicistronic reporter construct, pCAT-N-CLuc, and assessed translational activities in a cell-free coupled transcription-translation system. Luciferase is translated from the second cistron of the transcripts derived from these plasmids, following internal entry of ribosomes on the 5'NTR sequence that is located within the intercistronic space. In contrast, CAT is translated by a ribosome scanning of the upstream cistron and serves as a control for transcription efficiency (see Materials and Methods).
We first introduced a series of mutations into the putative helical segment (nt 44 to 47 and 115 to 118) that is located at the base of the domain II structure (Fig. 2A). This helical segment (which was predicted on the basis of a comparative analysis of the HCV and GBV-B sequences) (9) is common to the two predicted structures, as it was used to constrain the MFOLD prediction shown in Fig. 1B. Plasmid p45GA contained a CU-to-GA substitution involving nt 45 and 46 that should destabilize the helix, while p116UC contained a AG-to-UC substitution within the complementary strand of the helix, at nt 116 and 117. Plasmid p45GA/116UC contained both sets of mutations, potentially allowing reconstitution of the predicted secondary structure (Fig. 2A). Autoradiography of the products of translation demonstrated significant decreases in the quantities of luciferase expressed from p45GA and p116UC compared with pCAT-N-CLuc, despite relatively constant quantities of the CAT product (Fig. 2B; compare lanes 2 and 3 with lane 1). In contrast, there was little if any decrease in luciferase expressed from p45GA/116UC.
|
E1, p116UC-CE1, and
p45GA/116UC-CE1) within the background of a dicistronic plasmid which contains as its second cistron the entire HCV capsid-coding sequence and part of the E1 sequence. The relative translational activities of these constructs were similar to those of pCAT-N-CLuc and
its related mutant constructs in coupled transcription-translation reactions (data not shown). Thus, the nature of the second cistron did
not influence the impairment of IRES activity imposed by these mutations.
To assess the translational activities of the mutated 5'NTR sequences
in vivo, each of the plasmids depicted in Fig. 2A was transfected into
Huh-T7 cells shortly after infection with the recombinant vaccinia
virus vTF7-3 (17). CAT and luciferase activities were
determined in lysates prepared 24 h following transfection. When
relative luciferase/CAT ratios were calculated as a measure of IRES
strength in these cells, the translational activities of the single
mutants, p45GA and p116UC, were 50 and 23%, respectively, of that of
the wild type, pCAT-N-CLuc (Fig. 2C, bottom). However, the IRES
activity of the double mutant, p45GA/116UC, was equivalent to that of
the wild type (104%). These in vivo results are remarkably similar to
those obtained in the cell-free system in vitro.
These results confirm that the predicted helical segment involving nt
44 to 47 and 115 to 118 is required for optimal expression of IRES
activity. However, they also show that substantial IRES activity is
retained when the helix has been disrupted by the substitution of two
nucleotides. To determine whether more extensive substitutions within
this helix would completely extinguish IRES activity, we constructed
plasmids in which four of the putative base pairs were altered (Fig.
2A). Thus, p44GGAC contained a CCUG-to-GGAC substitution at nt 44 to
47, while p115GUCC contained a GUCC substitution involving the
complementary bases at nt 115 to 118. Each of these four-base mutants
had approximately 50% of the IRES activity of the wild-type sequence,
both in vivo and in vitro (data not shown). Again, the inclusion of
both mutations in p44GGAC/115GUCC allowed restoration of the predicted
helical segment, as it resulted in essentially normal translational
activity (data not shown). These additional results thus confirm those
obtained with the first set of mutants involving two base substitutions
(Fig. 2). They indicate that the helical segment predicted at the base
of the extended domain II stem-loop structure is required for optimal translational activity, but they confirm that disruption of this helix
does not completely eliminate IRES activity. This could be due to local
alternative base pair arrangements that are permissive for translation
or to retention of essential IRES structure due to the presence of the
four-base extension of this helical segment that is predicted within
the structure shown in Fig. 1B.
Nuclease analysis of RNA secondary structure within domain II. Previous work has shown that cobra venom nuclease V1 cleaves synthetic 5'NTR transcripts in the region from nt 116 to 118 (2). In addition, there are strong primer extension stops at nt 45 and at nt 118 and 119 in the absence of nuclease digestion (Fig. 3, lane 1). Since nuclease V1 preferentially cleaves double-stranded or helically stacked RNA substrates (13), these data are consistent with the base pairing predicted between nt 44 to 47 and nt 115 to 118. However, to confirm that these findings are due to these specific base pair interactions, RNA transcripts derived from pCAT-N-CLuc and its related mutants, p45GA, p116UC, and p45GA/116UC, were digested with RNases V1 and S1 and then used as templates in primer extension reactions.
|
Additional mutations which destabilize base pairing between nt 65 to 70 and nt 97 to 102 abolish IRES-directed translation. We next examined the predicted base pairing between nt 65 to 70 and nt 97 to 102, a helical segment that is also common to the two structures shown in Fig. 1. Mutations at nt 65 to 69 and/or nt 98 to 102 were created in the backgrounds of p45GA and p116UC, respectively (Fig. 4A). Thus, in addition to the CU-to-GA substitutions at nt 45 and 46, p45GA-65GUGAG contained a CACGC to GUGAG substitution at nt 65 to 69 that disrupts the second helical segment of the predicted structure. This mutated 5'NTR sequence demonstrated only 8.3% of the wild-type IRES activity in the cell-free translation system (Fig. 4B). Similarly, p116UC-98CGCAC, which had a GAGUG-to-CGCAC substitution at nt 98 to 102 as well as the AG-to-UC substitution at nt 116 and 117 (Fig. 4A), demonstrated only 5.7% of the wild-type IRES activity (Fig. 4B). The combination of these mutations in p45GA-65GUGAG/116UC-98CGCAC allowed reconstitution of the predicted secondary structure despite extensive primary sequence differences and restored the efficiency of IRES-directed translation in vitro to 32% of the wild-type level (Fig. 4A and B).
|
Secondary structure involving nt 50 to 64 and nt 103 to 114. The preceding results support the presence of helical segments that are predicted to be present in both of the structures shown in Fig. 1, but they do not distinguish between these two alternative foldings of the RNA. In an initial step to address this issue, we determined that the sequence comprising the large bulge loop located near the base of domain II in the structure shown in Fig. 1A is required for IRES activity. RNA transcripts with a deletion spanning nt 103 to 114 (pD103-114 [Fig. 4A]) demonstrated negligible IRES activity (Fig. 4B), indicating that retention of this sequence is essential for the translation.
Additional mutations were then constructed to evaluate potential base pairing in this region (Fig. 5A). In both of the predicted structures shown in Fig. 1, nt 50 to 52 are involved in base pair interactions leading to short helical segments. However, in the model shown in Fig. 1A, nt 50 to 53 pair with nt 61 to 64 to form the stem of hairpin IIa, while in the alternate model (Fig. 1B), nt 49 to 53 pair with nt 111 to 114 to form an extended helix joining the ends of domain II. Consistent with either prediction, a AGG-to-UUC substitution at nt 50 to 52 reduced translation to 32% of the wild-type level in the cell-free translation system and to 7.5% of the wild-type level in transfected BT7-H cells (Fig. 5B and C).
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
A considerable body of evidence supports the existence of the secondary and tertiary structures that have been proposed previously for the 3' half (domains III and IV) of the HCV 5'NTR. These data include the results of extensive comparative sequence analyses, the probing of secondary structure by nucleases or chemicals, and mutational analyses of IRES function (2, 9, 24, 27-29). Of these various approaches to determination of RNA structure, the most powerful has proven to be phylogenetic searches for covariant nucleotide substitutions that are indicative of conserved helical elements (2, 27). The results of such analyses allow critical constraints to be entered into computer-based RNA folding programs and permit rationally designed mutational studies. However, until the sequencing of GBV-B, it was difficult to apply this strategy to domain II of the HCV 5'NTR because of the absence of related sequences with an appropriate degree of nucleotide sequence divergence.
The recent discovery of GBV-B (23) provided such a sequence and has allowed a more accurate prediction of the domain II structure of HCV. This segment of the 5'NTR was predicted to form a complex structure spanning nt 44 to 117 and containing two hairpin loops (stem-loops IIa and IIb), a single large, internal bulge loop comprising nt 103 to 114, and several smaller internal bulge loops (Fig. 1A) (9). The existence of stem-loop IIa was supported by previous nuclease probing studies (2, 9). However, subsequent thermodynamic analyses suggested that a more stable structure could be formed by the collapse of the nucleotides involved in the putative IIa hairpin into the main stem structure (Fig. 1B). The mutational analysis described in this communication supports the existence of this latter structure and demonstrates that it is essential for efficient functioning of the HCV IRES.
These data are important, since an appreciation of the higher-ordered structures that contribute to the IRES is essential to understanding the mechanisms by which this RNA element controls cap-independent viral translation. Moreover, such data will be helpful in designing small-molecule inhibitors of IRES function that could become useful antiviral therapeutics. The analysis of the domain II structure reported here allows a more complete picture of the secondary structure of the entire HCV IRES (Fig. 6).
|
The data summarized in Fig. 2, 3, and 5 confirm the existence of two of the predicted helical segments that form the core of this structure. Mutations in plasmids p45GA and p116UC destabilized the helix at the base of the domain and resulted in a decrease in IRES-directed translation to about 50 and 20% of the wild-type (pCAT-N-CLuc) level. In contrast, p45GA/116UC, which contains both mutations, demonstrated nearly normal IRES activity. This reflects the restoration of the helical segment through base pairing of the mutated residues (Fig. 2A) and indicates that the helical structure is much more important for IRES activity than the primary nucleotide sequence within these segments of the RNA. Similar results were obtained in vitro and in vivo, with larger, 4-nt substitutions within this helical segment (data not shown) and with substitutions involving nt 50 to 52 and 111 to 113 (Fig. 5). Additional support for the basal helix of domain II comes from the analysis of V1 nuclease cleavage sites near the substitutions in these mutants. Remarkably, nucleotide substitutions within one strand of the helix affected V1 cleavage activity on the opposing strand as well (Fig. 3).
The results obtained in these experiments and the model shown in Fig.
1B are in general agreement with previous nuclease mapping of the
secondary structure of domain II, as are the results of V1 nuclease
mapping shown in Fig. 3. The major exception to this statement is the
cleavage between nt 57 and 60 that we observed previously using
nucleases with preferences for single-stranded substrates
(2). This finding suggests that the short helical segment
that is present between the two lower internal bulge loops in the model
shown in Fig. 1B either may not form or may be subject to breathing. It
is worth noting that this helical segment contributes little to the
folded energy of the proposed domain II structure. The calculated free
energies of the structure shown in Fig. 1B are 16.8 kcal/mol with and
15.5 kcal/mol without these base pair interactions. Two prominent
primer extension stops that occurred in the absence of V1 nuclease
cleavage (at nt 75 to 78 and 88 to 90 [Fig. 3]) are located within
the apical helical segment of the proposed structure. These stops most
likely result from the proposed secondary structure.
The 5'NTRs of the pestiviruses BVDV and HoCV, as well as the novel flavivirus GBV-B, also contain IRES elements. These IRESs contain structures that are remarkably similar to the domain III structure of HCV, including the centrally located pseudoknot that is present in HCV (18, 28). Following identification of the elements of the domain II structure that are conserved in HCV and GBV-B (9), we examined the pestivirus sequences for the possible presence of similar conserved structures within analogous regions of the 5'NTR. This search led to identification of putative structures within the BVDV and HoCV RNAs which are somewhat simpler than that present in HCV or GBV-B but which nonetheless share a number of characteristics with the HCV structure (Fig. 7). The most notable feature of these proposed domain II structures is the conservation of primary nucleotide sequences within the terminal loop and internal bulge loops (highlighted in Fig. 7), despite extensive variation within sequences that contribute to helical segments of the structure. The highly conserved nature of these loop sequences among flaviviruses that infect very different host species and diverse target tissues is remarkable. It suggests the possibility that these sequences interact with highly conserved elements of the host translational apparatus.
|
The extensive nucleotide substitutions in plasmids p45GA-65GUGAG and p116UC-98CGCAC almost completely abrogated translation directed by the HCV IRES, in vitro and in vivo (Fig. 4). On the other hand, p45GA-65GUGAG/116UC-98CGCAC, which contains a combination of these mutations, demonstrated 32 to 50% of the translational activity of the wild-type construct (Fig. 4B). This impressive restoration of IRES activity in the face of extensive changes in the primary nucleotide sequence of domain II reinforces the importance of the domain II structure to translation. However, the failure of p45GA-65GUGAG/116UC-98CGCAC to demonstrate translational activity that is equivalent to that of the wild-type IRES also indicates that the primary sequence within the internal helix is important for translation. It is tempting to relate this to the noncanonical A-G base pair within the internal helix of stem-loop IIb. An identical A-G base pair is located within the analogous helical segments of BVDV and HoCV (Fig. 7). Indeed, the sequences of this base-paired helical segment are remarkably conserved among these viruses. The conservation of this noncanonical base pair in these diverse viruses suggests that it may have functional importance. The double mutant p45GA-65GUGAG/116UC-98CGCAC contains a G-A base pair within the reconstituted helix, but in orientation opposite that of the A-G pair in the wild-type sequence. A-G/G-A base pairs may form through four different types of hydrogen bond interactions, each with a unique structure (26). Thus, the inversion of the A-G base pair may well have altered the conformation of the helix, creating either a change in the orientation of the apical segment of the stem-loop or a bubble within the stem itself. This may account for the less than complete restoration of translational activity observed with the double mutant.
Data from other laboratories support the importance of the terminal helical segment of stem-loop II. Rijnbrand et al. (19) and Reynolds et al. (15) investigated the role of the AUG triplet that is located within this stem-loop at nt 85 to 87. Nucleotide substitutions within this triplet led to loss of IRES activity. However, substitutions of the G at nt 87 were tolerated, provided that a compensatory substitution preserving the terminal helical segment was made at nt 79 (15).
Pestova et al. (14) recently demonstrated that the HCV and pestiviral IRESs are able to form binary complexes with purified 40S ribosome subunits in the absence of any canonical or noncanonical translation factors. Eucaryotic initiation factor 3 (eIF3) contributes to this process by binding both domain III of the IRES and the 40S subunit. This distinguishes these RNA elements from the picornaviral IRESs that form 48S complexes only in the presence of eIF2, eIF3, and eIF4. A reasonable hypothesis is that domain II of the HCV and pestiviral IRESs contributes to the process of ribosome subunit recognition through specific interactions of its conserved single-stranded loop segments with ribosome proteins or unpaired segments of the 18S RNA.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported in part by grants from the National Institute of Allergy and Immunology (U19-AI40035 and RO1-AI32599) and the Advanced Technology Program of the Texas Higher Education Coordinating Board.
We are grateful to R. Rijnbrand for comments and helpful criticism and to T. Zuker for making the MFOLD program available.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, 4.104 Medical Research Building, 301 University Blvd., Galveston, TX 77555-1019. Phone: (409) 772-2324. Fax: (409) 772-3757. E-mail: smlemon{at}utmb.edu.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
| 1. |
Ali, N., and A. Siddiqui.
1997.
The La antigen binds 5' noncoding region of the hepatitis C virus RNA in the context of the initiator AUG codon and stimulates internal ribosome entry site-mediated translation.
Proc. Natl. Acad. Sci. USA
94:2249-2254 |
| 2. |
Brown, E. A.,
H. Zhang,
L. H. Ping, and S. M. Lemon.
1992.
Secondary structure of the 5' nontranslated regions of hepatitis C virus and pestivirus genomic RNAs.
Nucleic Acids Res.
20:5041-5045 |
| 3. |
Choo, Q. L.,
G. Kuo,
A. J. Weiner,
L. R. Overby,
D. W. Bradley, and M. Houghton.
1989.
Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome.
Science
244:359-362 |
| 4. |
Davidson, F.,
P. Simmonds,
J. C. Ferguson,
L. M. Jarvis,
B. C. Dow,
E. A. Follett,
C. R. Seed,
T. Krusius,
C. Lin,
G. A. Medgyesi,
H. Kiyokawa,
G. Olim,
G. Duraisamy,
T. Cuypers,
A. A. Saeed,
D. Teo,
J. Conradie,
M. C. Kew,
M. Lin,
C. Nuchaprayoon,
O. K. Ndimbie, and P. L. Yap.
1995.
Survey of major genotypes and subtypes of hepatitis C virus using RFLP of sequences amplified from the 5' non-coding region.
J. Gen. Virol.
76:1197-1204 |
| 5. |
Fuerst, T.,
E. G. Niles,
F. W. Studier, and B. Moss.
1986.
Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase.
Proc. Natl. Acad. Sci. USA
83:8122-8126 |
| 6. | Fukushi, S., K. Katayama, C. Kurihara, N. Ishiyama, F. B. Hoshino, T. Ando, and A. Oya. 1994. Complete 5' noncoding region is necessary for the efficient internal initiation of hepatitis C virus RNA. Biochem. Biophys. Res. Commun. 199:425-432[Medline]. |
| 7. | Hayashi, N., H. Higashi, K. Kaminaka, H. Sugimoto, M. Esumi, K. Komatsu, K. Hayashi, M. Sugitani, K. Suzuki, O. Tadao, C. Nozaki, K. Mizuno, and T. Shikata. 1993. Molecular cloning and heterogeneity of the human hepatitis C virus (HCV) genome. J. Hepatol. 17:S94-S107. |
| 8. | Honda, M., G. Abell, D. Kim, and S. M. Lemon. Natural variation in translational activities of the 5' nontranslated RNAs of genotypes 1a and 1b hepatitis C virus: evidence for a long range RNA-RNA interaction outside of the internal ribosomal entry site. Submitted for publication. |
| 9. | Honda, M., E. A. Brown, and S. M. Lemon. 1996. Stability of a stem-loop involving the initiator AUG controls the efficiency of internal initiation of translation on hepatitis C virus RNA. RNA 2:955-968[Abstract]. |
| 10. | Honda, M., S. Kaneko, A. Sakai, M. Unoura, S. Murakami, and K. Kobayashi. 1994. Degree of diversity of hepatitis C virus quasispecies and progression of liver disease. Hepatology 20:1144-1151[Medline]. |
| 11. | Honda, M., L.-H. Ping, R. Rijnbrand, E. Amphlett, B. Clarke, D. Rowlands, and S. M. Lemon. 1996. Structural requirements for initiation of translation by internal ribosome entry within genome-length hepatitis C virus RNA. Virology 222:31-42[Medline]. |
| 12. | Kiyosawa, K., T. Sodeyama, E. Tanaka, Y. Gibo, K. Yoshizawa, Y. Nakano, S. Furuta, Y. Akahane, K. Nishioka, R. H. Purcell, and H. J. Alter. 1990. Interrelationship of blood transfusion, non-A, non-B hepatitis and hepatocellular carcinoma: analysis by detection of antibody to hepatitis C virus. Hepatology 12:671-675[Medline]. |
| 13. |
Lowman, H. B., and D. E. Draper.
1986.
On the recognition of helical RNA by cobra venom V1 nuclease.
J. Biol. Chem.
261:5396-5403 |
| 14. |
Pestova, T. V.,
I. N. Shatsky,
S. P. Fletcher,
R. J. Jackson, and C. U. Hellen.
1998.
A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs.
Genes Dev.
12:67-83 |
| 15. | Reynolds, J. E., A. Kaminski, A. R. Carroll, B. E. Clarke, D. J. Rowlands, and R. J. Jackson. 1996. Internal initiation of translation of hepatitis C virus RNA: the ribosome entry site is at the authentic initiation codon. RNA 2:867-878[Abstract]. |
| 16. | Reynolds, J. E., A. Kaminski, H. Kettinen, A. R. Carroll, D. J. Rowlands, and R. J. Jackson. 1995. Unique features of internal initiation of hepatitis C virus RNA translation. EMBO J. 14:6016-6020. |
| 17. | Rijnbrand, R., P. Bredenbeek, T. van der Straaten, L. Whetter, G. Inchauspe, S. Lemon, and W. Spaan. 1995. Almost the entire 5' non-translated region of hepatitis C virus is required for cap-independent translation. FEBS Lett. 365:115-119[Medline]. |
| 18. | Rijnbrand, R., T. van der Stratten, P. A. van Rijn, W. J. M. Spaan, and P. J. Bredenbeek. 1997. Internal entry of ribosomes is directed by the 5' noncoding region of classical swine fever virus and is dependent on the presence of RNA pseudoknot upstream of the initiation codon. J. Virol. 71:451-457[Abstract]. |
| 19. | Rijnbrand, R. C., T. E. Abbink, P. C. Haasnoot, W. J. Spaan, and P. J. Bredenbeek. 1996. The influence of AUG codons in the hepatitis C virus 5' nontranslated region on translation and mapping of the translation initiation window. Virology 226:47-56[Medline]. |
| 20. | Schultz, D. E., M. Honda, L. E. Whetter, K. L. McKnight, and S. M. Lemon. 1996. Mutations within the 5' nontranslated RNA of cell culture-adapted hepatitis A virus which enhance cap-independent translation in cultured African green monkey kidney cells. J. Virol. 70:1041-1049[Abstract]. |
| 21. |
Simmonds, P.,
E. C. Holmes,
T. A. Cha,
S. W. Chan,
F. McOmish,
B. Irvine,
E. Beall,
P. L. Yap,
J. Kolberg, and M. S. Urdea.
1993.
Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS-5 region.
J. Gen. Virol.
74:2391-2399 |
| 22. | Simons, J. N., S. M. Desai, D. E. Schultz, S. M. Lemon, and I. K. Mushahwar. 1996. Translation initiation in GB viruses A and C: evidence for internal ribosome entry and implications on genome organization. J. Virol. 70:6126-6135[Abstract]. |
| 23. |
Simons, J. N.,
T. J. Pilot-Matias,
T. P. Leary,
G. J. Dawson,
S. M. Desai,
G. G. Schlauder,
A. S. Muerhoff,
J. C. Erker,
S. L. Buijk,
M. L. Chalmers,
C. L. Van Sant, and I. K. Mushahwar.
1995.
Identification of two flavivirus-like genomes in the GB hepatitis agent.
Proc. Natl. Acad. Sci. USA
92:3401-3405 |
| 24. |
Smith, D. B.,
J. Mellor,
L. M. Jarvis,
F. Davidson,
J. Kolberg,
M. Urdea,
P.-L. Yap,
P. Simmonds, and International HCV Collaborative Study Group.
1995.
Variation of the hepatitis C virus 5' non-coding region: implications for secondary structure, virus detection and typing.
J. Gen. Virol.
76:1749-1761 |
| 25. | Strauss, J. H., E. G. Strauss, C. S. Hahn, and C. M. Rice. 1987. The genomes of alphaviruses and flaviviruses: organization and translation, p. 75-104. In D. J. Rowlands, M. A. Mayo, and B. W. J. Mahy (ed.), The molecular biology of the positive strand RNA viruses. Academic Press, London, England. |
| 26. | Tinoco, I. 1993. Structure of base pairs involving at least two hydrogen bonds, p. 603-7. In R. F. Gesteland, and J. F. Atkins (ed.), The RNA world. Cold Spring Harbor Press, Cold Spring Harbor, N.Y. |
| 27. |
Tsukiyama, K. K.,
N. Iizuka,
M. Kohara, and A. Nomoto.
1992.
Internal ribosome entry site within hepatitis C virus RNA.
J. Virol.
66:1476-1483 |
| 28. | Wang, C., S. Y. Le, N. Ali, and A. Siddiqui. 1995. An RNA pseudoknot is an essential structural element of the internal ribosome entry site located within the hepatitis C virus 5' noncoding region. RNA 1:526-537[Abstract]. |
| 29. |
Wang, C.,
P. Sarnow, and A. Siddiqui.
1994.
A conserved helical element is essential for internal initiation of translation of hepatitis C virus RNA.
J. Virol.
68:7301-7307 |
| 30. |
Wang, C.,
P. Sarnow, and A. Siddiqui.
1993.
Translation of human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism.
J. Virol.
67:3338-3344 |
| 31. |
Weiner, A. J.,
H. M. Geysen,
C. Christopherson,
J. E. Hall,
T. J. Mason,
G. Saracco,
F. Bonino,
K. Crawford,
C. D. Marion,
K. A. Crawford, et al.
1992.
Evidence for immune selection of hepatitis C virus (HCV) putative envelope glycoprotein variants: potential role in chronic HCV infections.
Proc. Natl. Acad. Sci. USA
89:3468-3472 |
| 32. |
Whetter, L. E.,
S. P. Day,
O. Elroy-Stein,
E. A. Brown, and S. M. Lemon.
1994.
Low efficiency of the 5' nontranslated region of hepatitis A virus RNA in directing cap-independent translation in permissive monkey kidney cells.
J. Virol.
68:5253-5263 |
Journal of Virology, February 1999, p. 1165-1174, Vol. 73, No. 2
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Friebe, P., Bartenschlager, R.
(2009). Role of RNA Structures in Genome Terminal Sequences of the Hepatitis C Virus for Replication and Assembly. J. Virol.
83: 11989-11995
[Abstract] [Full Text] -
Eng, F. J., Walewski, J. L., Klepper, A. L., Fishman, S. L., Desai, S. M., McMullan, L. K., Evans, M. J., Rice, C. M., Branch, A. D.
(2009). Internal Initiation Stimulates Production of p8 Minicore, a Member of a Newly Discovered Family of Hepatitis C Virus Core Protein Isoforms. J. Virol.
83: 3104-3114
[Abstract] [Full Text] -
Babaylova, E., Graifer, D., Malygin, A., Stahl, J., Shatsky, I., Karpova, G.
(2009). Positioning of subdomain IIId and apical loop of domain II of the hepatitis C IRES on the human 40S ribosome. Nucleic Acids Res
37: 1141-1151
[Abstract] [Full Text] -
Baugh, C., Shaohui Wang, , Bin Li, , Appleman, J. R., Thompson, P. A.
(2009). SCAN TM--A High-Throughput Assay for Detecting Small Molecule Binding to RNA Targets. J Biomol Screen
14: 219-229
[Abstract] -
Jaffrelo, L., Chabas, S., Reigadas, S., Pflieger, A., Wychowski, C., Rumi, J., Ventura, M., Toulme, J.-J., Staedel, C.
(2008). A functional selection of viral genetic elements in cultured cells to identify hepatitis C virus RNA translation inhibitors. Nucleic Acids Res
36: e95-e95
[Abstract] [Full Text] -
Gastaminza, P., Cheng, G., Wieland, S., Zhong, J., Liao, W., Chisari, F. V.
(2008). Cellular Determinants of Hepatitis C Virus Assembly, Maturation, Degradation, and Secretion. J. Virol.
82: 2120-2129
[Abstract] [Full Text] -
Masante, C., Mahias, K., Lourenco, S., Dumas, E., Cahour, A., Trimoulet, P., Fleury, H., Astier-Gin, T., Ventura, M.
(2008). Seven nucleotide changes characteristic of the hepatitis C virus genotype 3 5' untranslated region: correlation with reduced in vitro replication. J. Gen. Virol.
89: 212-221
[Abstract] [Full Text] -
Binder, M., Quinkert, D., Bochkarova, O., Klein, R., Kezmic, N., Bartenschlager, R., Lohmann, V.
(2007). Identification of Determinants Involved in Initiation of Hepatitis C Virus RNA Synthesis by Using Intergenotypic Replicase Chimeras. J. Virol.
81: 5270-5283
[Abstract] [Full Text] -
Kanda, T., Steele, R., Ray, R., Ray, R. B.
(2007). Small Interfering RNA Targeted to Hepatitis C Virus 5' Nontranslated Region Exerts Potent Antiviral Effect. J. Virol.
81: 669-676
[Abstract] [Full Text] -
Gastaminza, P., Kapadia, S. B., Chisari, F. V.
(2006). Differential Biophysical Properties of Infectious Intracellular and Secreted Hepatitis C Virus Particles. J. Virol.
80: 11074-11081
[Abstract] [Full Text] -
Baird, S. D., Turcotte, M., Korneluk, R. G., Holcik, M.
(2006). Searching for IRES. RNA
12: 1755-1785
[Abstract] [Full Text] -
Chard, L. S., Kaku, Y., Jones, B., Nayak, A., Belsham, G. J.
(2006). Functional Analyses of RNA Structures Shared between the Internal Ribosome Entry Sites of Hepatitis C Virus and the Picornavirus Porcine Teschovirus 1 Talfan. J. Virol.
80: 1271-1279
[Abstract] [Full Text] -
LESCOUTE, A., WESTHOF, E.
(2006). Topology of three-way junctions in folded RNAs.. RNA
12: 83-93
[Abstract] [Full Text] -
Muerhoff, A. S., Leary, T. P., Sathar, M. A., Dawson, G. J., Desai, S. M.
(2005). African origin of GB virus C determined by phylogenetic analysis of a complete genotype 5 genome from South Africa. J. Gen. Virol.
86: 1729-1735
[Abstract] [Full Text] -
Boni, S., Lavergne, J.-P., Boulant, S., Cahour, A.
(2005). Hepatitis C Virus Core Protein Acts as a trans-Modulating Factor on Internal Translation Initiation of the Viral RNA. J. Biol. Chem.
280: 17737-17748
[Abstract] [Full Text] -
Baril, M., Brakier-Gingras, L.
(2005). Translation of the F protein of hepatitis C virus is initiated at a non-AUG codon in a +1 reading frame relative to the polyprotein. Nucleic Acids Res
33: 1474-1486
[Abstract] [Full Text] -
Piron, M., Beguiristain, N., Nadal, A., Martinez-Salas, E., Gomez, J.
(2005). Characterizing the function and structural organization of the 5' tRNA-like motif within the hepatitis C virus quasispecies. Nucleic Acids Res
33: 1487-1502
[Abstract] [Full Text] -
Kikuchi, K., Umehara, T., Fukuda, K., Kuno, A., Hasegawa, T., Nishikawa, S.
(2005). A hepatitis C virus (HCV) internal ribosome entry site (IRES) domain III-IV-targeted aptamer inhibits translation by binding to an apical loop of domain IIId. Nucleic Acids Res
33: 683-692
[Abstract] [Full Text] -
Forton, D. M., Karayiannis, P., Mahmud, N., Taylor-Robinson, S. D., Thomas, H. C.
(2004). Identification of Unique Hepatitis C Virus Quasispecies in the Central Nervous System and Comparative Analysis of Internal Translational Efficiency of Brain, Liver, and Serum Variants. J. Virol.
78: 5170-5183
[Abstract] [Full Text] -
Pisarev, A. V., Chard, L. S., Kaku, Y., Johns, H. L., Shatsky, I. N., Belsham, G. J.
(2004). Functional and Structural Similarities between the Internal Ribosome Entry Sites of Hepatitis C Virus and Porcine Teschovirus, a Picornavirus. J. Virol.
78: 4487-4497
[Abstract] [Full Text] -
Kronke, J., Kittler, R., Buchholz, F., Windisch, M. P., Pietschmann, T., Bartenschlager, R., Frese, M.
(2004). Alternative Approaches for Efficient Inhibition of Hepatitis C Virus RNA Replication by Small Interfering RNAs. J. Virol.
78: 3436-3446
[Abstract] [Full Text] -
Reusken, C. B. E. M., Dalebout, T. J., Eerligh, P., Bredenbeek, P. J., Spaan, W. J. M.
(2003). Analysis of hepatitis C virus/classical swine fever virus chimeric 5'NTRs: sequences within the hepatitis C virus IRES are required for viral RNA replication. J. Gen. Virol.
84: 1761-1769
[Abstract] [Full Text] -
Beales, L. P., Holzenburg, A., Rowlands, D. J.
(2003). Viral Internal Ribosome Entry Site Structures Segregate into Two Distinct Morphologies. J. Virol.
77: 6574-6579
[Abstract] [Full Text] -
Yi, M., Lemon, S. M.
(2003). 3' Nontranslated RNA Signals Required for Replication of Hepatitis C Virus RNA. J. Virol.
77: 3557-3568
[Abstract] [Full Text] -
Sarnow, P.
(2003). Viral Internal Ribosome Entry Site Elements: Novel Ribosome-RNA Complexes and Roles in Viral Pathogenesis. J. Virol.
77: 2801-2806
[Full Text] -
Dumas, E., Staedel, C., Colombat, M., Reigadas, S., Chabas, S., Astier-Gin, T., Cahour, A., Litvak, S., Ventura, M.
(2003). A promoter activity is present in the DNA sequence corresponding to the hepatitis C virus 5' UTR. Nucleic Acids Res
31: 1275-1281
[Abstract] [Full Text] -
Tallet-Lopez, B., Aldaz-Carroll, L., Chabas, S., Dausse, E., Staedel, C., Toulme, J.-J.
(2003). Antisense oligonucleotides targeted to the domain IIId of the hepatitis C virus IRES compete with 40S ribosomal subunit binding and prevent in vitro translation. Nucleic Acids Res
31: 734-742
[Abstract] [Full Text] -
Laporte, J., Bain, C., Maurel, P., Inchauspe, G., Agut, H., Cahour, A.
(2003). Differential distribution and internal translation efficiency of hepatitis C virus quasispecies present in dendritic and liver cells. Blood
101: 52-57
[Abstract] [Full Text] -
Lanford, R. E., Guerra, B., Lee, H., Averett, D. R., Pfeiffer, B., Chavez, D., Notvall, L., Bigger, C.
(2002). Antiviral Effect and Virus-Host Interactions in Response to Alpha Interferon, Gamma Interferon, Poly(I)-Poly(C), Tumor Necrosis Factor Alpha, and Ribavirin in Hepatitis C Virus Subgenomic Replicons. J. Virol.
77: 1092-1104
[Abstract] [Full Text] -
Le, S.-Y., Zhang, K., Maizel, J. V. Jr
(2002). RNA molecules with structure dependent functions are uniquely folded. Nucleic Acids Res
30: 3574-3582
[Abstract] [Full Text] -
Kashiwagi, T., Hara, K., Kohara, M., Iwahashi, J., Hamada, N., Honda-Yoshino, H., Toyoda, T.
(2002). Promoter/Origin Structure of the Complementary Strand of Hepatitis C Virus Genome. J. Biol. Chem.
277: 28700-28705
[Abstract] [Full Text] -
Lafuente, E., Ramos, R., Martinez-Salas, E.
(2002). Long-range RNA-RNA interactions between distant regions of the hepatitis C virus internal ribosome entry site element. J. Gen. Virol.
83: 1113-1121
[Abstract] [Full Text] -
Fletcher, S. P., Jackson, R. J.
(2002). Pestivirus Internal Ribosome Entry Site (IRES) Structure and Function: Elements in the 5' Untranslated Region Important for IRES Function. J. Virol.
76: 5024-5033
[Abstract] [Full Text] -
Friebe, P., Lohmann, V., Krieger, N., Bartenschlager, R.
(2001). Sequences in the 5' Nontranslated Region of Hepatitis C Virus Required for RNA Replication. J. Virol.
75: 12047-12057
[Abstract] [Full Text] -
Odreman-Macchioli, F., Baralle, F. E., Buratti, E.
(2001). Mutational Analysis of the Different Bulge Regions of Hepatitis C Virus Domain II and Their Influence on Internal Ribosome Entry Site Translational Ability. J. Biol. Chem.
276: 41648-41655
[Abstract] [Full Text] -
Lytle, J. R., Wu, L., Robertson, H. D.
(2001). The Ribosome Binding Site of Hepatitis C Virus mRNA. J. Virol.
75: 7629-7636
[Abstract] [Full Text] -
Hellen, C. U.T., Sarnow, P.
(2001). Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev.
15: 1593-1612
[Full Text] -
Lyons, A. J., Lytle, J. R., Gomez, J., Robertson, H. D.
(2001). Hepatitis C virus internal ribosome entry site RNA contains a tertiary structural element in a functional domain of stem-loop II. Nucleic Acids Res
29: 2535-2541
[Abstract] [Full Text] -
Lanford, R. E., Lee, H., Chavez, D., Guerra, B., Brasky, K. M.
(2001). Infectious cDNA clone of the hepatitis C virus genotype 1 prototype sequence. J. Gen. Virol.
82: 1291-1297
[Abstract] [Full Text] -
Martínez-Salas, E., Ramos, R., Lafuente, E., López de Quinto, S.
(2001). Functional interactions in internal translation initiation directed by viral and cellular IRES elements. J. Gen. Virol.
82: 973-984
[Full Text] -
Zhao, W. D., Wimmer, E.
(2001). Genetic Analysis of a Poliovirus/Hepatitis C Virus Chimera: New Structure for Domain II of the Internal Ribosomal Entry Site of Hepatitis C Virus. J. Virol.
75: 3719-3730
[Abstract] [Full Text] -
Venkatesan, A., Dasgupta, A.
(2001). Novel Fluorescence-Based Screen To Identify Small Synthetic Internal Ribosome Entry Site Elements. Mol. Cell. Biol.
21: 2826-2837
[Abstract] [Full Text] -
Spahn, C. M. T., Kieft, J. S., Grassucci, R. A., Penczek, P. A., Zhou, K., Doudna, J. A., Frank, J.
(2001). Hepatitis C Virus IRES RNA-Induced Changes in the Conformation of the 40S Ribosomal Subunit. Science
291: 1959-1962
[Abstract] [Full Text] -
Kunkel, M., Lorinczi, M., Rijnbrand, R., Lemon, S. M., Watowich, S. J.
(2001). Self-Assembly of Nucleocapsid-Like Particles from Recombinant Hepatitis C Virus Core Protein. J. Virol.
75: 2119-2129
[Abstract] [Full Text] -
Banerjee, R., Dasgupta, A.
(2001). Specific Interaction of Hepatitis C Virus Protease/Helicase NS3 with the 3'-Terminal Sequences of Viral Positive- and Negative-Strand RNA. J. Virol.
75: 1708-1721
[Abstract] [Full Text] -
Jubin, R., Vantuno, N. E., Kieft, J. S., Murray, M. G., Doudna, J. A., Lau, J. Y. N., Baroudy, B. M.
(2000). Hepatitis C Virus Internal Ribosome Entry Site (IRES) Stem Loop IIId Contains a Phylogenetically Conserved GGG Triplet Essential for Translation and IRES Folding. J. Virol.
74: 10430-10437
[Abstract] [Full Text] -
Laporte, J., Malet, I., Andrieu, T., Thibault, V., Toulme, J.-J., Wychowski, C., Pawlotsky, J.-M., Huraux, J.-M., Agut, H., Cahour, A.
(2000). Comparative Analysis of Translation Efficiencies of Hepatitis C Virus 5' Untranslated Regions among Intraindividual Quasispecies Present in Chronic Infection: Opposite Behaviors Depending on Cell Type. J. Virol.
74: 10827-10833
[Abstract] [Full Text] -
Lerat, H., Shimizu, Y. K., Lemon, S. M.
(2000). Cell Type-Specific Enhancement of Hepatitis C Virus Internal Ribosome Entry Site-Directed Translation due to 5' Nontranslated Region Substitutions Selected during Passage of Virus in Lymphoblastoid Cells. J. Virol.
74: 7024-7031
[Abstract] [Full Text] -
Kolupaeva, V. G., Pestova, T. V., Hellen, C. U. T.
(2000). An Enzymatic Footprinting Analysis of the Interaction of 40S Ribosomal Subunits with the Internal Ribosomal Entry Site of Hepatitis C Virus. J. Virol.
74: 6242-6250
[Abstract] [Full Text] -
Gale, M. Jr., Tan, S.-L., Katze, M. G.
(2000). Translational Control of Viral Gene Expression in Eukaryotes. Microbiol. Mol. Biol. Rev.
64: 239-280
[Abstract] [Full Text] -
Odreman-Macchioli, F. E., Tisminetzky, S. G., Zotti, M., Baralle, F. E., Buratti, E.
(2000). Influence of correct secondary and tertiary RNA folding on the binding of cellular factors to the HCV IRES. Nucleic Acids Res
28: 875-885
[Abstract] [Full Text] -
Rijnbrand, R., Abell, G., Lemon, S. M.
(2000). Mutational Analysis of the GB Virus B Internal Ribosome Entry Site. J. Virol.
74: 773-783
[Abstract] [Full Text] -
Chang, M., Marquardt, A. P., Wood, B. L., Williams, O., Cotler, S. J., Taylor, S. L., Carithers, R. L. Jr., Gretch, D. R.
(2000). In Situ Distribution of Hepatitis C Virus Replicative-Intermediate RNA in Hepatic Tissue and Its Correlation with Liver Disease. J. Virol.
74: 944-955
[Abstract] [Full Text] -
Sbardellati, A., Scarselli, E., Tomei, L., Kekulé, A. S., Traboni, C.
(1999). Identification of a Novel Sequence at the 3' End of the GB Virus B Genome. J. Virol.
73: 10546-10550
[Abstract] [Full Text] -
Honda, M., Rijnbrand, R., Abell, G., Kim, D., Lemon, S. M.
(1999). Natural Variation in Translational Activities of the 5' Nontranslated RNAs of Hepatitis C Virus Genotypes 1a and 1b: Evidence for a Long-Range RNA-RNA Interaction outside of the Internal Ribosomal Entry Site. J. Virol.
73: 4941-4951
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»